High-throughput ligand profile characterization in novel cell lines expressing seven heterologous insect olfactory receptors for the detection of volatile plant biomarkers

Agriculturally important crop plants emit a multitude of volatile organic compounds (VOCs), which are excellent indicators of their health status and their interactions with pathogens and pests. In this study, we have developed a novel cellular olfactory panel for detecting fungal pathogen-related VOCs we had identified in the field, as well as during controlled inoculations of several crop plants. The olfactory panel consists of seven stable HEK293 cell lines each expressing a functional Drosophila olfactory receptor as a biosensing element along with GCaMP6, a fluorescent calcium indicator protein. An automated 384-well microplate reader was used to characterize the olfactory receptor cell lines for their sensitivity to reference VOCs. Subsequently, we profiled a set of 66 VOCs on all cell lines, covering a concentration range from 1 to 100 μM. Results showed that 49 VOCs (74.2%) elicited a response in at least one olfactory receptor cell line. Some VOCs activated the cell lines even at nanomolar (ppb) concentrations. The interaction profiles obtained here will support the development of biosensors for agricultural applications. Additionally, the olfactory receptor proteins can be purified from these cell lines with sufficient yields for further processing, such as structure determination or integration with sensor devices.


Generation of the stable OR cell lines
In vivo measured ligand profile data derived from the DoOR database (http:// neuro.uni-konst anz.de/ DoOR/ defau lt.html) 19,20 were used to select 12 ORs, which can be appropriate to detect the pathogen-related VOCs relevant for our study.To assemble a novel cell-based olfactory panel, 11 stable cell lines were successfully generated which expressed different Drosophila ORs and the fluorescent calcium ion indicator protein GCaMP6 21 in HEK293 cells.Out of these cell lines, seven were responsive to the respective OR-specific VOCs.Using these functional lines we constructed an olfactory assay in which binding of the examined odorants can be quantified by a fluorescence signal.
Four different proteins were expressed in each of the cell lines: the respective ORs, the ORCO universal coreceptor, the mCherry fluorescent protein as a marker for OR expression, and the GCaMP6 fluorescent calcium ion indicator protein.To ensure stable and high-level expression, bacterial artificial chromosome (BAC) vectors containing an approximately 70-kb long open chromatin region (BAC Rosa26 ) were used to deliver the transgenes into the cells.
In comparison to conventional expression vectors, the use of a full-length, 210 kb BAC Rosa26 expression vector could increase up to nine-fold the yield of heterologous secreted proteins 22 .It also helped to express high amount of recombinant sodium channels in CHO cells 23 .However, transfection of this large DNA was much less efficient compared to shorter plasmids.Therefore we generated a truncated 70 kb version of BAC Rosa26 by homologous recombination and FLP recombinase mediated mutagenesis.(Supplementary Fig. 4).
Transfection efficiency and long-term protein production capacity were tested for this truncated 70 kb BAC Rosa26 by GFP expression.Chinese hamster ovary (CHO) cells were nucleoporated with a plasmid, original 210 kb BAC Rosa26 and truncated 70 kb BAC Rosa26 vector all containing CAG promoter, GFP and Neomycin resistance gene as a selection marker.The ratio of GFP positive cells were approximately 3-times higher 24 h post transfection for truncated 70 kb BAC Rosa26 transfected cells (12% GFP positive of living cells) than for original 210 kb BAC Rosa26 transfected ones (4.1%) while it was less compared to plasmid transfected cells (17.1%) (Supplementary Fig. 4).The same cell pools were cultured for an additional 2 months in the presence of antibiotic selection pressure.Both BAC Rosa26 transfected cell pools showed high GFP intensity levels (Supplementary Fig. 4) with 88.3% GFP positive cells for the original BAC Rosa26 and 99.3% of the truncated BAC Rosa26 while in the plasmid transfected cell pool only 42.7% of the cells showed GFP fluorescence despite of constant selection pressure.
As the truncated 70 kb BAC Rosa26 could ensure high protein expression level but combined with more efficient transfection compared to the original version we used this as an expression vector in this study.
The expression of the OR and mCherry proteins was driven by the strong and constitutive CAG promoter 24 .The remaining two proteins, ORCO and GCaMP6, were expressed as a single fusion protein by linking GCaMP6 N-terminally to ORCO to allow membrane localization.Based on the experience of Corcoran et al. 25 constitutive ORCO expression can lead to cell death after several weeks.In order to ensure long-term viability of our OR cell lines, the doxycycline-inducible Ptet-T6 promoter 26 was chosen to drive the expression of the GCaMP6-ORCO fusion protein.
To test if the arrangement of genetic elements has an impact on gene expression or not, we cloned and transfected them in two different formats.In the OR13a, OR47a, OR85b, and OR98a cell lines the two expression units were physically integrated tandem (Fig. 1A) into BAC Rosa26 .In this arrangement the Ptet-T6 promoter exhibited no baseline expression.Based on binding experiments with the ORCO-activating artificial ligand VUAA1 27 there was no GCaMP6-ORCO expression without doxycycline treatment whereas a strong induction was recorded 48 h after doxycycline treatment (Supplementary Fig. 1A).The OR10a, OR49b, and OR71a cell lines, on the other hand, were generated by cotransfection of the same two expression units (Fig. 1A).
In these cell lines, the Ptet-T6 promoter had a substantial baseline activity which was further increased upon doxycycline induction (Supplementary Fig. S1B).In the light of these results, we concluded that tandem integration of the expression units is the method to choose for future cell lines as it prevented uninduced Ptet-T6 promoter activity.Nevertheless, this leaky promoter activity did not hinder the maintenance of these cell lines as their responsiveness was stable during the 60 days monitored (Supplementary Fig. S2).Thus, the previous observation that constitutive ORCO expression was detrimental to long-term cell culture 25 could not be confirmed for Drosophila OR10a, OR49b, and OR71a + ORCO combinations in our experiments.The OR cell lines may have different sensitivity to constitutive ORCO expression because the ion permeability of the channel depends on the OR identity 8 .Alternatively, components in the culture medium may activate the ORs in some cell lines, but not in others, due to their different ligand profiles.In four stable cell lines (OR7a, OR19a, OR69a, and OR47b) none of the tested VOCs could activate the ORs despite the fact that OR7a, OR19a and OR69a showed strong responses to multiple VOCs according to the DoOR database.Similarly to the seven responding cell lines, the mCherry fluorescence signal was strongly visible indicating the presence of at least the OR transcripts as mCherry was transcribed from the same mRNA as the ORs.All cell lines responded to VUAA1 indicating the presence of functional GCaMP6-ORCO fusion proteins.Lack of response of some ORs in heterologous expression systems was reported in several previous studies 17,[28][29][30][31][32] It was shown that translation can be a bottleneck in these cases.Functional OR protein expression can be improved by codon optimization of OR genes instead of using wild type sequences 33 .In our study, we used a codon-optimized version of ORCO, however, we did not optimize any of the OR genes.OR release from the ER and trafficking to the plasma membrane could have been hindered in non-responding cell lines, too.The use of signal peptide tags might help in these cell lines as it was previously shown that their use could significantly enhance the intensity of the Ca 2+ response of the Drodophila OR47a/ ORCO transfected cells in transient expression studies 34 .
Out of the seven responsive OR cell lines reported here only one, OR13a was stably expressed so far in an insect cell line (Sf21) 35 .This and other ORs were expressed transiently either by mRNA injection into Xenopus oocytes or by transfection into immortalized cell lines: OR13a 36 , OR10a and OR71a 37,38 , OR47a 9,34,39 , OR49b 39,40 , and OR85b 41,42 .In contrast, all our stable cell lines can be used for months for direct measurements.Moreover, the cell cultures can be scaled up, then cryopreserved and thawed again at any time for subsequent measurements.

The responsiveness of the OR cell lines to their reference ligands
The functionality of the OR cell lines was verified by the artificial ORCO agonist VUAA1 and at least one ORspecific odorant (Fig. 2).To identify OR-specific reference ligands, two or three strong ligand candidates were chosen based on the DoOR database (http:// neuro.uni-konst anz.de/ DoOR/ defau lt.html) 20 and tested at 100 μM final concentration.The compound that triggered the highest response was chosen as the reference ligand for the given OR.For four cell lines (OR49b, OR71a, OR85b, and OR98a)-from our plant disease-related ligand list-the strongest agonists were not the ones which were suggested by the DoOR database.We used styrene instead of 2-methylphenol for OR49b, 6-methyl-5-hepten-2-one instead of 4-ethylguaiacol for OR71a and instead of ethyl benzoate for OR98a, and 2-heptanone instead of butyl acetate for OR85b as reference ligands (Fig. 2B, Supplementary Table 2).
We used the GFP-linked calcium ion indicator GCaMP6, in order to measure the response of the cells as a fluorescence signal (Fig. 1B, C).
The maximum ∆F values measured on the OR cell lines were exceptionally high, ranging between 124 and 462 ΔF% in response to 50 μM VUAA1 and 29-339 ΔF% to their reference ligands during long term stability testing (Supplementary Fig. S2), in contrast to 20-60% previously measured in comparable experimental systems 25,35,43 .OR expression level of the cells can show some variation between measurement days.To minimize any VOC response differences due to this variation, the measured ∆F values were normalized to average cell line-specific ∆F values in response to 50 μM VUAA1 measured on the same day and were counted as 1.These VUAA1 normalized ∆F values were used to compare the different receptors and ligands.
We tested for the origin of the Ca 2+ ions on two cell lines, OR47a and (GCaMP6-GSG)ORCO, with assay buffer lacking Ca 2+ and assay buffer lacking Ca 2+ and containing EGTA.Neither of the cell lines responded to the ORCO agonist ligand (Supplementary Fig. S3), indicating that the signal is caused by extracellular Ca 2+ .
The time requirement for the measurement of one VOC sample in the seven OR cell lines was 13 min when measured in triplicates (three wells/sample/cell line).It was possible to measure 16 samples within approximately 3.5 h in a whole 384-well microplate.www.nature.com/scientificreports/

Comparison of ligand sensitivity with previously published OR-expressing cell lines
The concentration-dependent effect of the reference ligands on their cognate cell lines was examined in more detail.Twelve different ligand concentrations were measured on each cell line in an overall 0.01 μM to 100 μM concentration range with 5 repetitions with three technical replicates each time.The half-maximal effective concentration (EC50), and the maximal response compared to 50 μM VUAA1 were calculated (Fig. 2B).
Based on these parameters, a comparative evaluation of the OR cell lines was performed.There are a number of studies in which HEK293 cells have been used for functional characterization of insect ORs 25,33,34,[44][45][46] .In these studies, typical EC50 values and ligand sensitivity of the ORs were very similar to our results, but our system shows a much higher fluorescence intensity change (ΔF) in response to VOC ligands.
The sensitivity of the OR47a to pentyl acetate varied between different studies.In Xenopus oocytes, a range of 50-300 μM could trigger receptor responses 9 , while in a similar system the calculated EC50 was 10.7 ± 2.0 µM 39 and 30.8 ± 1.28 µM 34 .In our OR47a cell line, the EC50 was 2.22 μM for pentyl acetate (Fig. 2B), which indicates about five times higher sensitivity.
For the OR49b cell line, we used styrene as a reference ligand (see "Plant disease-related VOC measurements on the OR cell lines".below) but measured the EC50 for 2-methylphenol (o-cresol), too.With 13.95 μM (Supplementary file Data summary.xlsx) of EC50 our OR49b cell line was 17-times more sensitive to this ligand than previously measured in Xenopus oocytes in which the calculated EC50 was 239 ± 76 µM 39 .In a recent study, the same OR was expressed transiently in HEK293T cells, which reacted to 2-methylphenol above the limit of detection in a 2 μM to 800 μM concentration range.Though the EC50 was not calculated, 80 μM concentration triggered a 38.5% response 40 .
The OR85b receptor response to 2-heptanone was measured previously in Xenopus oocytes by Nichols and Luetje 42 with an EC50 of 70 ± 20 μM, and by Misawa et al. 41 with an average EC50 of 45.6 μM compared to the average 68.9 μM value we obtained (Fig. 2B).Once responsive OR cell lines were selected, we tested them for sensing fungal pathogens (powdery mildew, Botrytis cinerea, Fusarium spp., Pyrenophora spp., and gray mold) of important crop plants (wheat, barley, grape, lettuce, rape, spinach, and strawberry).To this end, we had compiled a pathogen-related VOC catalog primarily based on our GC-MS analyses of infected samples (published results 18 and unpublished data of our laboratory) and also systematically retrieved from published literature data [47][48][49][50] .Together with strong ligands for the seven ORs according to the DoOR database, a set of 66 compounds from our compilation was measured on all OR cell lines in 1 μM, 10 μM, and 100 μM concentrations (Fig. 3, Supplementary Table S2).

A
Among the 1386 VOC concentration-OR combinations measured, 156 (11%) gave positive odorant responses.As the reference ligand (6-methyl-5-hepten-2-one) with the lowest overall VUAA1-normalized response elicited a 0.2 VUAA1-normalized response (OR71a, Fig. 2), this value was taken as the threshold to separate strong/ medium reactions from low intensity ones.The majority (60.8%) of the responses were of low intensity, i.e., less than this 20% threshold of the VUAA1-normalized response.Reactions with strong (at least 40% of the VUAA1 response) or medium (20-40% of the VUAA1 response) intensity were recorded in 17% and 22.2% of all positive cases, respectively.
In 11 (2.4%) of the 462 VOC-OR combinations tested, the OR cell lines responded over the whole concentration range measured.High sensitivity to a ligand at the lowest concentration did not always guarantee the typical logistic relationship between ligand concentration and response intensity.Several odorants only activated ORs at the medium concentration, with moderate increase in response at higher concentrations.As an example, isopentyl acetate first activated the OR47a cell line at 10 μM and increasing the ligand concentration to 100 μM increased the response intensity from 12 to 18% (Supplementary Table S2).Some cell lines (and their ORs) showed a well-defined affinity for structurally similar compounds.For instance, OR49b was highly specific for aromatic compounds, as only these elicited strong or medium responses, with the exception of butyl acetate.Figure 3 summarizes the VOC ligand profiles of the OR cell lines and the chemical structures of strong and medium ligands.
We have a special focus on VOCs induced by wheat powdery mildew infections.Quantitative experimental data from consecutive years have therefore been collected in open field plots as well as from controlled inoculations of several cultivars.Six powdery mildew-specific volatile biomarkers have been identified and quantified in the headspace of healthy and diseased wheat plants 18 .Four of these biomarker VOCs were commercially available and three of them, 1-octen-3-ol, (5Z)-octa-1,5-dien-3-ol, and 3-octanone were in the detectable range of two OR cell lines in samples from diseased wheat plants (Table 1), while the concentration of 1-heptanol was below the detection limit of the OR13a cell line.

Comparison of the OR cell line ligand profiles with the DoOr database
The DoOr database-derived response profiles, which are collections of in vivo measured datasets, served as a good starting point for the identification of candidate ORs and the prediction of their response profiles to our VOC ligands.The DoOR database sets the scale by comparing the measured data to each other, while we took the maximum response value for each OR induced by VUAA1 as 1, and compared the effect of all other ligands to this.This essentially creates a database with a similar response profile ranging from 0 to 1, but this way of calculating response magnitudes is clearly different from the data in the DoOR database, therefore response magnitude values cannot be directly compared.Nevertheless, it is an important issue to investigate how much functional responses in an expression system correspond with in vivo data.
Twenty-two out of the 66 VOCs tested in the present study have not been tested in the DoOR database, so receptor responses to these VOCs represent new information.OR10a was the best-studied receptor for our ligand set; the DoOR database contained response values for 41 VOCs, while OR71a was the least studied with only 21 previously measured VOCs.The OR10a, OR47a, and OR49b cell lines responded at 100 μM to all VOCs that were predicted as a strong ligand (at least 0.4 response) for these ORs according to the DoOR database (5/5, 8/8 and 2/2, resp. in Supplementary Table S2).Similarly, a high matching rate (11/15, 73%) was also found for the OR98a cell line.However, for three cell lines, OR13a, OR85b and OR71a the respective success rates were only 57% (4/7), 30% (3/10), and 25%(1/4).These observations indicate the importance of testing the ligand profile and sensitivity in different expression systems, and emphasize the potential role of cellular environment in determining sensitivity profiles of individual ORs.

Concluding remarks
We generated responsive OR cell lines which stably express seven functional Drosophila ORs together with GCaMP6, a fluorescent calcium ion indicator protein.These cell lines were produced by transfection of HEK293 cells with a novel BAC expression vector carrying an open chromatin region to ensure higher and more stable expression levels in a long term.The OR cell lines detected odorant molecules from the ppb concentration in the liquid phase.The response was quick with the cells reaching maximum fluorescence intensity change within 10 s.Peaks of fluorescence intensity change were as high as 462% for the ORCO agonist VUAA1 and 339% for reference ligands, which is outstanding compared to the published data of ca.50%.The concentration-response profiles of the OR cell lines were mapped for 66 plant-and plant pathogen-derived VOCs over a concentration range of three orders of magnitude.We recorded ligand-specific fluorescence responses in 11% of all 1386 measured combinations of VOC concentrations and OR cell lines, similarly to the 17% described by Hallem et al. 17 .In terms of plant pathogen-related VOC sensing, three major volatile biomarkers specific for powdery mildew infection were in detectable concentration in diseased wheat samples 18 for the OR13a and OR47a cell lines.The OR proteins expressed here can be purified from the cells and coupled to other signal-transducing elements.For   www.nature.com/scientificreports/instance, systems like the ones developed earlier 37,51 may be utilized for our assay too, and such coupling steps could further improve sensitivity for the ligands explored here.

Molecular cloning of OR genes into plasmid and BAC expression vectors
The OR genes OR7a (FlyBase ID FBtr0071186), OR47a (FBtr0088111), and a codon-optimized version of ORCO (FBtr0113193) were synthesized (Thermo Fisher Scientific) as double-stranded linear gene fragments.The cDNAs of the OR10a, OR13a, OR19a, OR47b, OR49b, OR67b, OR69a, OR71a, OR85b, OR98a genes were cloned from the wild-type Canton S strain of D. melanogaster.Total RNA was extracted from adult flies by TRIzol Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions.Total RNA (1 μg) was treated with DNase I, then reverse transcribed with oligo(dT) 12-18 primers and SuperScript IV enzyme (Invitrogen).The OR genes were amplified with gene-specific primers containing an AscI recognition sequence on both primers (except SwaI for OR19a) and a Kozak consensus sequence on the forward primers (Supplementary Table S1).
The above 12 OR genes linked to a wild-type internal ribosome entry site from the encephalomyocarditis virus (EMCV IRES) and the mCherry fluorescent protein gene 52 were inserted into a modified pBlueScript KS (Agilent/Stratagene) called plasmid "L" 22 .The OR genes were inserted into this vector at the AscI (SwaI) restriction site, while IRES-mCherry at the PmeI restriction site and the resulting plasmids were named as MIO(receptor name)p.The N-terminus of ORCO was fused to GCaMP6 21 via a Gly 2 SerGly 3 SerGly linker.Subsequently, this fusion gene was cloned into a modified plasmid "L" at the AscI restriction site, in which the CAG promoter 24 was replaced by a Doxycycline-inducible Ptet-T6 bidirectional promoter 26 and its cognate transactivator protein (3G) gene (derived from AAVS1_Puro_Tet3G_3xFLAG_Twin_Strep, a gift from Yannick Doyon: Addgene plasmid #92099 53 .The resulting plasmid was named as DOGGp in this work.MIO13a/47a/85b/98aDOGGp plasmids were generated by ligating SalI-and SrfI-digested MIOp with Bsu36I-digested DOGGp (Fig. 1A).All enzymes were purchased from Thermo Fisher Scientific (except for SrfI, which was from New England Biolabs).Cloning was performed in the E. coli DH10B strain.All plasmid sequences are available upon request.
For the generation of the BAC expression vectors, a modified version of the murine Rosa26 BAC DNA (BAC PAC Resources Children's Hospital Oakland Research Institute clone number: RP24-85I15) was used as the vector backbone.Briefly, the original 210-kb BAC was shortened by removing approximately 70-70 kb DNA sequences from both ends of the genomic insert.First, two targeting constructs carrying 50 bp long homologous sequence regions (HR) on both ends (5′ targeting construct: HR-frt-Kanamycin-frt-HR, 3′ targeting constructs: HR-frt3-Ampicillin-frt3-HR) were generated by PCR (primers in Supplementary Table S1).The 5′ targeting construct was recombined into the targeted region replacing the original sequence by ET-cloning/recombineering 54,55 , then the cassette was excised by FLPe recombinase 56 .Subsequently, recombination and cassette excision were repeated with the 3′ targeting construct.
For ET-cloning DH10B E. coli harboring the original 210-kb BAC Rosa26 were electroporated with the temperature sensitive pSC101-BAD-gbaA (pRed/ET plasmid on Supplementary Fig. 4) plasmid which carries the recombinase proteins necessary for homologous recombination.Cells harboring the pSC101-BAD-gbaA plasmid were selected in tetracycline (5 μg/ml) at 30 °C overnight.Bacterial cells derived from one single positive colony were cultured overnight at 30 °C and transferred to 50 ml of fresh medium next day.At an optical density (OD600) of 0.2, the expression of the recombinogenic proteins was induced by the addition of l-arabinose (to a 0.3-0.4% final concentration) and by shifting the temperature to 37 °C.After one additional hour, cells were harvested and electrocompetent cells were prepared by a double wash with ice cold distilled water.Cells were resuspended in 10% ice cold glycerol solution and aliquoted to Eppendorf tubes (100 μl) prior snap freezing in liquid N 2 or directly used for electroporation.100 ng of linear DNA was electroporated to these cells with a Gene Pulser Xcell (BioRad) electroporator.Cells were incubated for 70 min at 37 °C without antibiotics in LB medium and plated on LB-agar containing the respective antibiotic (15 μg/ml kanamycin after 5′ targeting construct recombination and 100 μg/ml ampicillin after 3′ targeting construct recombination) at 37 °C overnight.
In the next step, bacterial cells derived from one single positive colony were cultured overnight at 37 °C in LB/ antibiotic (ampicillin/kanamycin).The next day electrocompetent cells were prepared the same way as for ETcloning and were electroporated with the expression plasmid 706 FLP which expresses the FLPe recombinase 56 to excise the antibiotic cassette flanked by frt/frt3 sites in the 5′/3′ targeting constructs.Cells were resuspended in LB medium without antibiotics after electroporation and incubated for 70 min at 30 °C. 100 μl of cell suspension was plated into LB-agar containing tetracycline (3 μg/ml) and the appropriate antibiotics for the BAC (chloramphenicol 12.5 µg/ml) and incubated overnight at 30 °C.The plate was incubated at 37 °C for 4-6 h. 10 single colonies were picked and streaked out to another LB agar/chloramphenicol plate from the left to the right, each in one row and cultured 37 °C overnight.Single colonies were picked from this plate and we plate them on https://doi.org/10.1038/s41598-023-47455-4

Figure 1 .
Figure 1.Generation and characterization of OR-expressing cell lines.(A) DNA vector constructs for OR cell line production: MIOp plasmid containing the corresponding OR and mCherry, and DOGGB BAC expression vector coding for the GCaMP6-Orco fusion protein were cotransfected to generate OR10a, OR49b and OR71a cell lines (upper panel); MIODOGGB BAC expression vectors containing the corresponding OR and all other components were used to generate OR13a, OR47a, OR85b and OR98a cell lines (lower panel).The inducible Ptet bidirectional promoter only activates GCaMP6-Orco protein expression upon doxycycline (Dox) treatment.(B) Detection mechanism of the cell lines: the OR + Orco receptor complex opens upon VOC ligand binding, the resulting Ca 2+ influx is detectable as GCaMP6-mediated fluorescence.VOC volatile organic compound.(C) Fluorescence signal intensity change of the OR47a cell line 48 h after doxycycline induction (orange curves) and without doxycycline (blue curves) in response to 50 µM VUAA1.Each curve represents a technical replicate measured in separate wells of a 384-well microplate.RFU relative fluorescence unit, F MAX maximal RFU value during the measurement, F 0 average baseline fluorescence, ∆F was calculated according to the equation shown.

Figure 2 .
Figure 2. Concentration-dependent response of the OR cell lines to the respective reference VOC ligands.(A) VUAA1-normalized response was calculated by dividing VOC response with the average of VUAA1 response on the day of the measurement for the corresponding OR cell line.Concentration-response curves are shown as the average of 5 measurements, error bars represent the standard error (SD).(B) EC50 values and maximal VUAA1 normalized responses were calculated from the fitted Hill equation,where the Hill coefficient was constrained to 1 and the maximal response was maximum 1.The calculations and the original traces are available in the supplement. https://doi.org/10.1038/s41598-023-47455-4www.nature.com/scientificreports/

Figure 3 .
Figure 3. VOC ligand profile of the OR cell lines.Each VOC was measured at 1, 10, and 100 µM final concentration on each receptor.VUAA1-normalized response was calculated as described in methods.Values are averages of at least three biological replicates for responsive combinations and two for the non-responsive combinations.The chemical structures of the strong and medium ligands are shown for each cell line.Heat map: white-green colors correspond to the lowest-highest values measured for each cell line.

Table 1 .
Concentration range and detectability of major wheat powdery mildew biomarker VOCs by two OR cell lines.